The present invention generally relates to a method for selectively transducing pathologic hyperproliferative mammalian cells in a heterogeneous cell preparation comprising retroviral-mediated transduction of the pathologic cell with a nucleic acid encoding a gene product having tumor suppressive function.
Throughout this application various publications are referenced within parentheses. The disclosures of these publications in their entireties are hereby incorporated by reference in this application in order to more fully describe the state of the art to which this invention pertains.
The human p53 gene encodes a 53 kilodalton nuclear phosphoprotein (Lane, D. P., et al., Genes and Dev., 4:1-8 (1990); Lee, Y-HP, Breast Cancer Res.and Trmt, 19:3-13 (1991); Rotter, V., et al., Adv. Can. Res., 57:257-72 (1991)). The p53 protein was first identified as a cellular protein in SV40-transformed cells that was tightly bound to the SV40 T antigen (Lane, D. P., et al. Nature, 278:261-3 (1979)). The wild type p53 gene has the characteristics of a tumor suppressor gene. It is similar to the prototype of tumor suppressor genes, the retinoblastoma gene (RB), in that loss of heterozygosity of the p53 or RB genes characterizes the phenotype of many types of tumor cells (Hollstein, M. et al., Science, 253:49-51 (1991); Levine, A. J., et al., Biochimica et Biophysica Acta, 1032:119-36 (1990); Levine, A. J., et al., Nature 351:453-6 (1991); Weinberg, R. A. Science, 254:1138-46 (1991)). In human malignancies associated with p53 alterations, this loss of heterozygosity usually results from the loss of one allele (allelic deletion), while the other allele suffers one or more somatic mutations. Unlike RB, however, certain mutations in the p53 gene are capable of immortalizing rodent cells and enhancing the tumorigenicity of established cell lines (Jenkins, J. R., et al., Nature, 312:651-4 (1984)). Mutant but not wild type p53 can cooperate with the activated ras oncogene in transforming primary rat embryo fibroblasts (Eliyahu, D., et al., Nature, 312(13):646-9 (1984); Parada, L. F., et al., Nature, 312:649-51 (1984)). Other events related to tumor progression also appear to be associated with the expression of mutant p53. Among these is differential modulation of the multiple drug resistance gene (MDR1) by wild type as compared to altered p53. In this case, mutant p53 specifically stimulates the MDR1 promoter, while wild type p53 exerts repression (Chin, K-V., et al., Science, 255:459-62 (1992)). Another possible way in which mutant p53 could promote tumorigenesis is by reducing tumor cell responsiveness to transforming growth factor-xcex2, a negative regulator of cell proliferation (Gerwin, B. I., et al., PNAS USA, 89:2759-63 (1992)).
In addition to the in vitro data described above two animal models have been described that implicate p53 in tumor formation. Transgenic mice expressing a mutant p53 gene display a high incidence of lung, bone and lymphoid tumors (Lavigueur, A., et al. Mol. Cell. Biol., 9(9):3982-91 (1989)). In addition, p53-null mice (Donehower, L. A., et al., Nature, 356(19):215-21 (1992)) show an increased risk of spontaneous neoplasms, the most frequently observed being malignant lymphoma.
Other data which support the conclusion that mutant p53 plays an important role in tumorigenesis include re-introduction of the wild type p53 gene into human tumor cell lines which lack p53 expression. In this case, wild type p53 can reverse the malignant phenotype as measured by colony formation in soft agar and tumor formation in nude mice (Chen, P. L., et al., Science, 250:1576-80 (1990); Cheng, J., et al., Can. Res., 52:222-6 (1992); Baker, S. J., et al., Science, 249:912-15 (1990); Isaacs, W. B., et al., Can. Res., 51:4716-20 (1991); Casey, G., et al., Oncogene, 6(10):1791-7 (1991); Shaw, P., et al., PNAS USA, 89:4495-99 (1992); Takahashi, T., et al., Can. Res., 52:2340-3 (1992)). Tumor cell types which have shown conversion of a non-malignant phenotype as a result of the introduction of wild type p53 expression include prostate (Isaacs, W. B., et al., supra), breast (Casey, G. et al. supra), colon (Baker, S J., et al., supra; Shaw, P. et al., supra) lung (Takahashi, T. et al., supra), and lymphoblastic leukemia (Cheng, J. et al., supra). Other data suggest that introduction of wild type p53 into tumor cells which have lost endogenous p53 expression appears to be cytotoxic (Johnson, P. et al., Mol. Cell. Biol., 11(1):1-11 (1991)). In some cases the re-introduction of wild type p53 may result in programmed cell death, or apoptosis (Yonish-Rouach, E. et al., Nature, 352:345-7 (1991)). The work described above indicates strongly that alteration of the wild type p53 gene has a role in multiple aspects of tumorigenesis and that reintroduction of the wild type p53 coding sequence can have a negative regulatory function or cytotoxic effect on malignant cells.
Clinical data suggest that inactivating mutations in the p53 gene are among the most common types of mutations associated with human malignancy (Rotter, V. et al. supra; Nigro, J. M. et al., Nature, 342:705-8 (1989); Gaidano, G. et al., PNAS USA, 88:5413-7 (1991); Cheng, J. et al., Mol. Cell. Biol., 10(10):5502-09 (1990)). A classical example is the Li-Fraumeni syndrome, a familial syndrome of several neoplasms, including breast cancer, sarcomas and others. Specific mutations in the p53 gene are found in affected members of the family and appear to be associated with the predisposition to develop early cancers (Malkin, D. et al., Science, 250:1233 (1990); Srivastava, S. et al., Nature, 348:747 (1990)). Several laboratories have reported that alterations in the p53 gene accompany the evolution of human CML (chronic myelogenous leukemia) to blast crisis (acute phase) (Ahuja, H. et al., J.Clin.Invest., 87:2042-7 (1991); Foti, A. et al., Blood, 77(11):2441-4 (1991); Feinstein, E. et al., PNAS USA, 88:6293-7 (1991)). In one CML patient who reverted briefly from the acute phase to a second chronic phase, the inactivating point mutation in p53 which appeared concomitantly with the acute phase disappeared and the wild type sequence re-emerged (Foti, A. et al., supra). These data indicate that alterations which inactivate the tumor suppressive activity of p53 may represent pivotal events in the progression from the chronic to the acute phase of human CML. Other clinical data also suggest an important role for p53 mutations in disease progression. These include a number of hematologic neoplasms as well as solid tumors (Rotter, V. et al. supra; Ahuja, H. et al., J.Clin.Invest., supra; Ahuja, H. et al., PNAS USA, 86:6783-6787 (1989); Mori, N. et al., Br. J. of Haem., 81:235-240 (1992); Porter, P. L. et al., Am.J.Path., 140(1):145-53 1992)). Recent reports show a dramatic association between altered p53 and shortened survival in breast cancer (Thor, A. D. et al., J. Natl.Can. Inst., 84(11):845-55 (1992); Isola, J. et al., J.Natl. Can. Inst.,84(14):1109-14 (1992); Callahan, R. J.Natl. Can.Inst., 84:826-7 (1992)).
The present invention generally relates to a method for selectively transducing pathologic hyperproliferative mammalian cells comprising retroviral-mediated transduction of pathologic cells with a nucleic acid encoding a gene product having tumor suppressive function. The methodology provided involves the introduction of a stably expressed tumor suppressor gene into a heterogeneous cell preparation (containing both normal and pathologic hyperproliferative cells) and, under suitable conditions, selectively transducing phenotypically pathologic hyperproliferative cells, suppressing the pathologic phenotype and reinfusing the treated cell preparation into the patient. Also provided by this invention is a method for treating a pathology in a subject caused by the absence of, or the presence of a pathologically mutated tumor suppressor gene.